SUMMARY Ultrasound (US) is used for a variety of therapeutic applications. Over a range of different frequencies and powers, US has been shown to produce to produce modest increases in arterial diameter and tissue perfusion in animal models of limb and myocardial ischemia. In the initial funding period for this award, we described how the combination of US with microbubble (MB) contrast agents that undergo inertial cavitation during high-power contrast-enhanced US (CEU) produces much greater augmentation of limb skeletal muscle perfusion (up to 10-fold) than US alone. Brief CEU cavitation protocols were found to reverse limb ischemia for >24 hrs in animal models, and a clinical trial in patients with peripheral artery disease (PAD) confirmed that MB cavitation increases limb perfusion by several fold. In the course of our studies, optimal conditions for these bioeffects were investigated which mandated us to design novel US pulse schemes and 3-D exposure capability. From a mechanistic standpoint, we carefully mapped pathways responsible for cavitation-induced flow augmentation which rely on shear-mediated ATP release from endothelial cells and erythrocytes, with secondary purinergic vasodilation through downstream mediators (NO, prostaglandins, adenosine). Knowledge of the optimal conditions and mechanistic underpinnings is critical for our current efforts to apply cavitation and activation of ATP channels to treat ischemic disease by augmenting flow or by other potentially beneficial anti-thrombotic and anti-inflammatory effects of purinergic signaling. The overall aim of this renewal is to leverage knowledge from the first funding period in order to explore the therapeutic role of cavitation and non-cavitation US for acute and chronic ischemic syndromes. In Aim 1 preclinical models will be used to determine whether limb flow-augmentation from MB cavitation using previously-optimized pulse schemes can: (a) prevent tissue necrosis in acute ischemia, with a particular focus on the effect of clinical variables (age, sex, hyperlipidemia, diabetes), and (b) improve wound healing and limb function in chronic disease. The functional role of purinergic vascular signaling will be evaluated by using inhibitor strategies or gene--modified models. In Aim 2 we will determine whether MB cavitation directly augments myocardial perfusion in acute MI using murine models that allow us to manipulate purinergic pathways, and in primate models that more closely resemble human biology. We will also study how US-mediated ATP release has the potential to mitigate inflammation, and microvascular thrombosis upon reperfusion. In Aim 3 we will test whether US energy from multi-element high-power intra- arterial catheters increases downstream perfusion through shear-mediated purinergic pathways. This Aim is based on evidence that therapeutic US catheters used in patients with pulmonary embolism can reduce pulmonary vascular resistance even without clot lysis. Our proposal represents the translational steps for development of non-invasive therapies for acute and chronic vascular diseases and will form the basis for the design of clinical trials that we plan to initiate as the key unsolved issues are addressed.